1. Field of the Invention
This invention relates to optical analysis of the contents of capillary tubes or other small separation channels.
2. Description of Related Art
Laser-induced fluorescence detection is an important optical detection technique for the analysis of biological samples, such as macromolecules that are subjected to capillary electrophoresis. Because capillary tube dimensions and laser detection volumes are extremely small in the capillary tube fluorescence context, however, proper optical alignment of the capillary tube detection volume with respect to the fluorescence excitation source is critical. Maximum sensitivity is achieved only when the entire optical train is properly aligned. Typically, the excitation source is focused into a narrow band of .ltoreq.100 microns, which illuminates a small detection region of the capillary tube. Not only is it difficult to correctly position the capillary tube in the excitation source light path, but it is also difficult to maintain this position once it is achieved. This is due in part to shifting of the tube under the stress of high field strength or thermal variations in the environment. It is also a result of the requirement that capillary tubes be readily replaceable.
A common method for accomplishing alignment entails filling the capillary tube with a fluorescent solution and adjusting the tube-and-beam intersection while monitoring the fluorescence with a detector, such as a photomultiplier tube or a photodiode to achieve a fluorescence intensity maximum. This technique is only relevant for open-tube applications, however, such as capillary zone electrophoresis, since the fluorescent solution is not easily removed from gel-filled tubes that will be used for electrophoresis of samples. Even so, it is generally undesirable to insert fluorescent material into any separation tube for alignment or other control purposes, because adsorption of fluorescent material residue on the tube walls may interfere with the analysis of samples. Furthermore, this method does not allow for dynamic alignment during analysis.
U.S. Pat. No. 5,208,466, commonly assigned, discloses an apparatus and method for aligning a capillary tube with respect to a laser beam source for fluorescence detection. That invention employs photosensors to detect the light reflection and scatter patterns originating from the walls of the capillary tube and utilizes the symmetry of those scatter patterns to indicate optimum alignment. The photosensors may also provide a means of feedback control.
It is an object of the present invention to provide a simple, precise means for alignment of a capillary tube's contents with a detection optics path, both in static and fully-automatable dynamic modes. For economic purposes, the invention should employ a minimal number of detectors. It is a further object to effect alignment without the need to introduce fluorescent material into the capillary tube.